Risk-based prioritization of suspects detected in riverine water using complementary chromatographic techniques.

Surface waters are widely used as drinking water sources and hence their quality needs to be continuously monitored. However, current routine monitoring programs are not comprehensive as they generally cover only a limited number of known pollutants and emerging contaminants. This study presents a risk-based approach combining suspect and non-target screening (NTS) to help extend the coverage of current monitoring schemes. In particular, the coverage of NTS was widened by combining three complementary separations modes: Reverse phase (RP), Hydrophilic interaction liquid chromatography (HILIC) and Mixed-mode chromatography (MMC). Suspect lists used were compiled from databases of relevant substances of very high concern (e.g., SVHCs) and the concentration of detected suspects was evaluated based on ionization efficiency prediction. Results show that suspect candidates can be prioritized based on their potential risk (i.e., hazard and exposure) by combining ionization efficiency-based concentration estimation, in vitro toxicity data or, if not available, structural alerts and QSAR.based toxicity predictions. The acquired information shows that NTS analyses have the potential to complement target analyses, allowing to update and adapt current monitoring programs, ultimately leading to improved monitoring of drinking water sources.

Application of in vitro data in physiologically-based kinetic models for quantitative in vitro-in vivo extrapolation: A case-study for baclofen.

Physiologically-based kinetic (PBK) models can simulate concentrations of chemicals in tissues over time without animal experiments. Nevertheless, in vivo data are often used to parameterise PBK models. This study aims to illustrate that a combination of kinetic and dynamic readouts from in vitro assays can be used to parameterise PBK models simulating neurologically-active concentrations of xenobiotics. Baclofen, an intrathecally administered drug to treat spasticity, was used as a proof-of-principle xenobiotic. An in vitro blood-brain barrier (BBB) model was used to determine the BBB permeability of baclofen needed to simulate plasma and cerebrospinal concentrations. Simulated baclofen concentrations in individuals and populations of adults and children generally fall within 2-fold of measured clinical study concentrations. Further, in vitro micro-electrode array recordings were used to determine the effect of baclofen on neuronal activity (cell signalling). Using quantitative in vitro-in vivo extrapolations (QIVIVE) corresponding doses of baclofen were estimated. QIVIVE showed that up to 4600 times lower intrathecal doses than oral and intravenous doses induce comparable neurological effects. Most simulated doses were in the range of administered doses. This show that PBK models predict concentrations in the central nervous system for various routes of administration accurately without the need for additional in vivo data.

Hyperthermia exacerbates the acute effects of psychoactive substances on neuronal activity measured using microelectrode arrays (MEAs) in rat primary cortical cultures in vitro.

Hyperthermia is a well-known, potentially life-threatening, side effect of stimulant psychoactive substances that worsens the neurological outcome of hospitalized patients. However, current in vitro methods to assess the hazard of psychoactive substances do not account for hyperthermia. Therefore, this study determined the potency of five psychoactive substances (cocaine, MDMA (3,4-methylenedioxymethamphetamine), methamphetamine, 3-MMC (3-methylmethcathinone) and TFMPP (3-trifluoromethylphenylpiperazine)) to affect neuronal activity at physiological and hyperthermic conditions. Neuronal activity of rat cortical cultures grown on microelectrode arrays (MEAs) was recorded at 37 °C before exposure. Following 30 min and 4.5 h drug exposure (1-1000 µM) at 37 °C or 41 °C, neuronal activity was measured at either 37 °C or 41 °C. Without drug exposure, hyperthermia induced a modest decrease in neuronal activity. Following acute (30 min) exposure at 37 °C, all drugs concentration-dependently inhibited neuronal activity. Increasing the temperature to 41 °C significantly exacerbated the reduction of neuronal activity ~ 2-fold for all drugs compared to 37 °C. Prolonged (4.5 h) exposure at 41 °C decreased neuronal activity comparable to 37 °C. Neuronal activity (partly) recovered following drug exposure at both temperatures, although recovery from exposure at 41 °C was less pronounced for most drugs. None of the exposure conditions affected viability. Since acute exposure at hyperthermic conditions exacerbates the decrease in neuronal activity induced by psychoactive substances, effects of hyperthermia should be included in future hazard assessment of illicit drugs and new psychoactive substances (NPS).

Hazard Characterization of Synthetic Cathinones Using Viability, Monoamine Reuptake, and Neuronal Activity Assays.

Synthetic cathinones are the second largest class of new psychoactive substances (NPS) on the drug market. Despite the large number of different cathinones and their abundant use, hazard characterization is mainly limited to their potential to inhibit monoamine transporters. To expand the current hazard characterization, we first investigated the acute effects of several synthetic cathinones [4-methylethcathinone (4-MEC), 3-methylmethcathinone (3-MMC), 4-MMC, methylone, pentedrone, α-pyrrolidinovalerophenone (α-PVP), and 3,4-methylenedioxypyrovalerone (MDPV)] on human dopamine, norepinephrine, and serotonin reuptake transporters (hDAT, hNET, and hSERT), which were stably transfected in human embryonic kidney (HEK) 293 cells. Next, we examined effects on spontaneous neuronal activity in rat primary cortical cultures grown on microelectrode arrays (MEAs) as an integrated endpoint for neurotoxicity. Changes in neuronal activity were assessed after acute (30 min) and prolonged (4.5 h) exposure. Moreover, we investigated whether neuronal activity recovered after washout of the exposure (24 h after the start of the 5 h exposure). Low micromolar concentrations of synthetic cathinones inhibited monoamine uptake via hDAT and hNET, while higher cathinone concentrations were needed to inhibit uptake via hSERT. Comparable high concentrations were needed to inhibit spontaneous neuronal activity during acute (30 min) and prolonged (4.5 h) exposure. Notably, while the inhibition of neuronal activity was reversible at low concentrations, only partial recovery was seen following high, but non-cytotoxic, concentrations of synthetic cathinones. Synthetic cathinones with either a pyrrolidine moiety or long alkyl-tail carbon chain more potently inhibit monoamine uptake via hDAT and neuronal activity. Monoamine uptake via hNET was most potently inhibited by synthetic cathinones with a pyrrolidine moiety. The combination of integrated measurements (MEA recordings of neuronal activity) with single target assays (monoamine reuptake transporter inhibition) indicates inhibition of hDAT and hNET as the primary mode of action of these synthetic cathinones. Changes in neuronal activity, indicative for additional mechanisms, were observed at higher concentrations.

Cardiotoxicity screening of illicit drugs and new psychoactive substances (NPS) in human iPSC-derived cardiomyocytes using microelectrode array (MEA) recordings.

The use of recreational drugs, including new psychoactive substances (NPS), is paralleled by emergency department visits of drug users with severe cardiotoxicity. Drug-induced cardiotoxicity can be the (secondary) result of increased norepinephrine blood concentrations, but data on potential drug-induced direct effects on cardiomyocyte function are scarce. The presence of hundreds of NPS therefore calls for efficient screening models to assess direct cardiotoxicity. We investigated effects of four reference compounds (3-30 nM dofetilide, nifedipine and isoproterenol, and 1-10 µM mexiletine) and six recreational drugs (0.01-100 µM cocaine, 0.01-1000 µM amphetamine, MDMA, 4-fluoroamphetamine, α-PVP and MDPV) on cardiomyocyte function (beat rate, spike amplitude and field potential duration (FPD ≈ QT interval in ECGs)), using Pluricyte® human-induced pluripotent stem cell (hiPSC)-derived cardiomyocytes cultured on ready-to-use CardioPlate™ multi-well microelectrode arrays (mwMEAs). Moreover, the effects of exposure to recreational drugs on cell viability were assessed. Effects of reference compounds were in accordance with the literature, indicating the presence of hERG potassium (dofetilide), sodium (mexiletine) and calcium (nifedipine) channels and α-adrenergic receptors (isoproterenol). All recreational drugs decreased the spike amplitude at 10-100 µM. All amphetamine-type stimulants and α-PVP decreased the beat rate at 300 µM, while cocaine and MDPV did so at 10 µM and 30 µM, respectively. All drugs increased the FPD, however at varying concentrations. MDMA, MDPV and amphetamine affected cardiomyocyte function at concentrations relevant for human exposure, while other drugs affected cardiomyocyte function only at higher concentrations (≥ 10 µM). Cell viability was only mildly affected at concentrations well above the lowest concentrations affecting cardiomyocyte function. We demonstrate that MEA recordings of hiPSC-derived cardiomyocytes enable screening for acute, direct effects on cardiomyocyte function. Our data further indicate that tachycardia in patients exposed to recreational drugs is likely due to indirect drug effects, while prolonged repolarization periods (prolonged QTc interval) could (partly) result from direct drug effects on cardiomyocyte function.

Changes in neuronal activity in rat primary cortical cultures induced by illicit drugs and new psychoactive substances (NPS) following prolonged exposure and washout to mimic human exposure scenarios.

The use of new psychoactive substances (NPS) is increasing despite associated health risks and limited pharmacological and toxicological knowledge. Information is available mainly for acute effects on specific targets like monoamine transporters and receptors. Recently, we have shown the ability of several NPS and illicit drugs to modulate neuronal activity during acute exposure. While these acute measurements provide valuable information regarding the potency and possible structure-activity relationships, an exposure scenario more representative of human exposure would increase insight and aid translation to the human situation. Therefore, we investigated the effects on neuronal activity after acute (30 min) and prolonged (5 h) exposure to amphetamine-type stimulants, cathinones, hallucinogens, piperazines and cocaine using rat primary cortical cultures grown on multi-well microelectrode arrays. To investigate the reversibility of effects, activity was also measured after a washout period of 19 h. During acute exposure, all compounds concentration-dependently decreased neuronal activity. Compared to acute exposure, prolonged exposure did not further decrease neuronal activity. Following washout, effects of 3 out of 11 drugs (methamphetamine, cocaine, and benzylpiperazine) were fully reversible, whereas effects induced by MDMA, PMMA and α-PVP were partially reversible. Neuronal activity did not recover after 19 h washout following exposure to the highest concentration of MDPV, 2C-B, 25B-NBOMe, and TFMPP. On the contrary, exposure to low concentrations of methylone, and to some extent of 2C-B, increased neuronal activity after the washout period. Hazard characterization of emerging NPS should include at least an acute exposure to determine a potency rank order. Supplementing the (acute and prolonged) exposure scenario with a washout period allows investigation of the reversibility of effects. The possibility of a neuronal network to regain activity after drug exposure appears independent of drug class or IC50 values for acute and prolonged exposure. Even though neuronal activity (partly) recovers after washout following exposure to most drugs, it is perturbing that complete recovery of neuronal activity is observed only for a minority of the tested drugs.

Differential effects of psychoactive substances on human wildtype and polymorphic T356M dopamine transporters (DAT).

Many psychoactive substances affect the human dopamine (DA) reuptake transporter (hDAT). Polymorphisms in the encoding gene could affect the functionality of the transporter and consequently alter effects of psychotropic and recreational drugs. Recently, a T356 M single nucleotide polymorphism in the human SLC6A3 gene was described, which resulted in functional impairments of DA uptake. Therefore, we investigated the effects of 10 psychoactive substances (0.01-1000 µM)) on DA uptake in human embryonic kidney (HEK) 293 cells transiently overexpressing wildtype (WT) or T356 M hDAT. Our data shows that T356 M hDAT has a 3 times lower Vmax and a 3 times higher Km compared to WT hDAT. Additionally, all psychoactive substances inhibited DA uptake by T356 M and WT hDAT. The DA reuptake inhibitors (methylphenidate, cocaine, and bupropion) inhibited DA uptake by WT hDAT most potently, followed by amphetamine-type stimulants [4-fluoroamphetamine (4-FA), amphetamine and MDMA], selective serotonin reuptake inhibitors (SSRI; fluoxetine and citalopram) and arylcyclohexylamines [methoxetamine (MXE) and ketamine]. Compared to DA uptake by WT hDAT, bupropion, methylphenidate, cocaine, and MXE less potently inhibited DA uptake by T356 M hDAT, while citalopram more potently inhibited uptake. The differences in IC50 values between T356 M and WT hDAT were considerable (3-45 fold). As such, the presence of this polymorphism could affect treatment efficiency with these substances as well as susceptibly for toxicity and addiction for individuals carrying this polymorphism.

Flotillins in the intercalated disc are potential modulators of cardiac excitability.

BACKGROUND: The intercalated disc (ID) is important for cardiac remodeling and has become a subject of intensive research efforts. However, as yet the composition of the ID has still not been conclusively resolved and the role of many proteins identified in the ID, like Flotillin-2, is often unknown. The Flotillin proteins are known to be involved in the stabilization of cadherins and desmosomes in the epidermis and upon cancer development. However, their role in the heart has so far not been investigated. Therefore, in this study, we aimed at identifying the role of Flotillin-1 and Flotillin-2 in the cardiac ID. METHODS: Location of Flotillins in human and murine cardiac tissue was evaluated by fluorescent immunolabeling and co-immunoprecipitation. In addition, the effect of Flotillin knockout (KO) on proteins of the ID and in electrical excitation and conduction was investigated in cardiac samples of wildtype (WT), Flotillin-1 KO, Flotilin-2 KO and Flotilin-1/2 double KO mice. Consequences of Flotillin knockdown (KD) on cardiac function were studied (patch clamp and Multi Electrode Array (MEA)) in neonatal rat cardiomyocytes (NRCMs) transfected with siRNAs against Flotillin-1 and/or Flotillin-2. RESULTS: First, we confirmed presence in the ID and mutual binding of Flotillin-1 and Flotillin-2 in murine and human cardiac tissue. Flotillin KO mice did not show cardiac fibrosis, nor hypertrophy or changes in expression of the desmosomal ID proteins. However, protein expression of the cardiac sodium channel NaV1.5 was significantly decreased in Flotillin-1 and Flotillin-1/2 KO mice compared to WT mice. In addition, sodium current density showed a significant decrease upon Flotillin-1/2 KD in NRCMs as compared to scrambled siRNA-transfected NRCMs. MEA recordings of Flotillin-2 KD NRCM cultures showed a significantly decreased spike amplitude and a tendency of a reduced spike slope when compared to control and scrambled siRNA-transfected cultures. CONCLUSIONS: In this study, we demonstrate the presence of Flotillin-1, in addition to Flotillin-2 in the cardiac ID. Our findings indicate a modulatory role of Flotillins on NaV1.5 expression at the ID, with potential consequences for cardiac excitation.

Neurotoxicity screening of new psychoactive substances (NPS): Effects on neuronal activity in rat cortical cultures using microelectrode arrays (MEA).

While the prevalence and the use of new psychoactive substances (NPS) is steadily increasing, data on pharmacological, toxicological and clinical effects is limited. Considering the large number of NPS available, there is a clear need for efficient in vitro screening techniques that capture multiple mechanisms of action. Neuronal cultures grown on multi-well microelectrode arrays (mwMEAs) have previously proven suitable for neurotoxicity screening of chemicals, pharmaceuticals and (illicit) drugs. We therefore used rat primary cortical cultures grown on mwMEA plates to investigate the effects of eight NPS (PMMA, α-PVP, methylone, MDPV, 2C-B, 25B-NBOMe, BZP and TFMPP) and two 'classic' illicit drugs (cocaine, methamphetamine) on spontaneous neuronal activity. All tested drugs rapidly and concentration-dependently decreased the weighted mean firing rate (wMFR) and the weighted mean burst rate (wMBR) during a 30 min acute exposure. Of the 'classic' drugs, cocaine most potently inhibited the wMFR (IC50 9.8 µM), whereas methamphetamine and the structurally-related NPS PMMA were much less potent (IC50 100 µM and IC50 112 µM, respectively). Of the cathinones, MDPV and α-PVP showed comparable IC50 values (29 µM and 21 µM, respectively), although methylone was 10-fold less potent (IC50 235 µM). Comparable 10-fold differences in potency were also observed between the hallucinogenic phenethylamines 2C-B (IC50 27 µM) and 25B-NBOMe (IC50 2.4 µM), and between the piperazine derivatives BZP (IC50 161 µM) and TFMPP (IC50 19 µM). All drugs also inhibited the wMBR and concentration-response curves for wMBR and wMFR were comparable. For most drugs, IC50 values are close to the estimated human brain concentrations following recreational doses of these drugs, highlighting the importance of this efficient in vitro screening approach for classification and prioritization of emerging NPS. Moreover, the wide range of IC50 values observed for these and previously tested drugs of abuse, both within and between different classes of NPS, indicates that additional investigation of structure-activity relationships could aid future risk assessment of emerging NPS.

Effect fingerprinting of new psychoactive substances (NPS): What can we learn from in vitro data?

The use of new psychoactive substances (NPS) is increasing and currently >600 NPS have been reported. However, limited information on neuropharmacological and toxicological effects of NPS is available, hampering risk characterization. We reviewed the literature on the in vitro neuronal modes of action to obtain effect fingerprints of different classes of illicit drugs and NPS. The most frequently reported NPS were selected for review: cathinones (MDPV, α-PVP, mephedrone, 4-MEC, pentedrone, methylone), cannabinoids (JWH-018), (hallucinogenic) phenethylamines (4-fluoroamphetamine, benzofurans (5-APB, 6-APB), 2C-B, NBOMes (25B-NBOMe, 25C-NBOMe, 25I-NBOMe)), arylcyclohexylamines (methoxetamine) and piperazine derivatives (mCPP, TFMPP, BZP). Our effect fingerprints highlight the main modes of action for the different NPS studied, including inhibition and/or reversal of monoamine reuptake transporters (cathinones and non-hallucinogenic phenethylamines), activation of 5-HT2receptors (hallucinogenic phenethylamines and piperazines), activation of cannabinoid receptors (cannabinoids) and inhibition of NDMA receptors (arylcyclohexylamines). Importantly, we identified additional targets by relating reported effect concentrations to the estimated human brain concentrations during recreational use. These additional targets include dopamine receptors, α- and ß-adrenergic receptors, GABAAreceptors and acetylcholine receptors, which may all contribute to the observed clinical symptoms following exposure. Additional data is needed as the number of NPS continues to increase. Also, the effect fingerprints we have obtained are still incomplete and suffer from a large variation in the reported effects and effect sizes. Dedicated in vitro screening batteries will aid in complementing specific effect fingerprints of NPS. These fingerprints can be implemented in the risk assessments of NPS that are necessary for eventual control measures to reduce Public Health risks.

Depsidones inhibit aromatase activity and tumor cell proliferation in a co-culture of human primary breast adipose fibroblasts and T47D breast tumor cells.

Naturally occurring depsidones from the marine fungus Aspergillus unguis are known to have substantial anti-cancer activity, but their mechanism of action remains elusive. The purpose of this study was to examine the anti-aromatase activity of two common depsidones, unguinol and aspergillusidone A, in a co-culture system of human primary breast adipose fibroblasts and hormonal responsive T47D breast tumor cells. Using this in vitro model it was shown that these depsidones inhibit the growth of T47D tumor cells most likely via inhibition of aromatase (CYP19) activity. The IC50 values of these depisidones were compared with the aromatase inhibitors letrozole and exemestane. Letrozole and exemestane had IC50 values of respectively, 0.19 and 0.14 µM, while those for Unguinol and Aspergillusidone A were respectively, 9.7 and 7.3 µM. Our results indicate that among the depsidones there maybe aromatase inhibitors with possible pharmacotherapeutical relevance.

Measuring inhibition of monoamine reuptake transporters by new psychoactive substances (NPS) in real-time using a high-throughput, fluorescence-based assay.

The prevalence and use of new psychoactive substances (NPS) is increasing and currently over 600 NPS exist. Many illicit drugs and NPS increase brain monoamine levels by inhibition and/or reversal of monoamine reuptake transporters (DAT, NET and SERT). This is often investigated using labor-intensive, radiometric endpoint measurements. We investigated the applicability of a novel and innovative assay that is based on a fluorescent monoamine mimicking substrate. DAT, NET or SERT-expressing human embryonic kidney (HEK293) cells were exposed to common drugs (cocaine, dl-amphetamine or MDMA), NPS (4-fluoroamphetamine, PMMA, α-PVP, 5-APB, 2C-B, 25B-NBOMe, 25I-NBOMe or methoxetamine) or the antidepressant fluoxetine. We demonstrate that this fluorescent microplate reader-based assay detects inhibition of different transporters by various drugs and discriminates between drugs. Most IC50 values were in line with previous results from radiometric assays and within estimated human brain concentrations. However, phenethylamines showed higher IC50 values on hSERT, possibly due to experimental differences. Compared to radiometric assays, this high-throughput fluorescent assay is uncomplicated, can measure at physiological conditions, requires no specific facilities and allows for kinetic measurements, enabling detection of transient effects. This assay is therefore a good alternative for radiometric assays to investigate effects of illicit drugs and NPS on monoamine reuptake transporters.